Blood-brain barrier permeability and increased levels of amino acids in cerebrospinal fluid are associated with brain alterations in rats with acute liver failure

Blood-brain barrier permeability and increased levels of amino acids in cerebrospinal fluid are associated with brain alterations in rats with acute liver failure | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Blood-brain barrier permeability and increased levels of amino acids in cerebrospinal fluid are associated with brain alterations in rats with acute liver failure Pedro Arend Guazzelli, Felipe dos Santos Fachim, Anderson Santos Travassos, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2093824/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Acute liver failure (ALF) is a life-threatening medical condition that often leads to hepatic encephalopathy (HE). Animals and humans with HE have shown elevated cerebrospinal fluid (CSF) levels of glutamine and glutamate, which may be associated with brain impairment. In this study, we aim to evaluate the relationship between blood-brain barrier (BBB) integrity and CSF amino acid levels with the neurological status of rats after subtotal hepatectomy. Adult male Wistar rats underwent a subtotal hepatectomy (removing 92% of hepatic mass or SHAM group) and were divided into 4 (four) cohorts. Animals with ALF presented severe neurological impairment and high mortality rates when compared to the SHAM group (Cohort 1). We performed a hepatic function test 24 hours after subtotal hepatectomy, which demonstrated a significant increase of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total Bilirubin, Direct Bilirubin, Prothrombin time and Ammonia levels in blood (Cohort 2). Additionally, the increase of amino acids, glutamine, and albumin levels in CSF as well as of Evans Blue penetration into the brain tissue was correlated with the neurological grades of HE, indicating signs of impaired BBB permeability induced by ALF (Cohort 3). Furthermore, the animals’ mortality rate showed a positive correlation with the increase of amino acid levels in CSF following subtotal hepatectomy (after 12h, Cohort 4). Our data highlight the potential role of amino acid levels in CSF, especially glutamine, for detection purposes even in the early stages of HE and suggest that these molecules offer prognostic value for patients progressing to coma and death. Acute liver failure Hepatic encephalopathy Cerebrospinal Fluid Amino Acids Albumin Blood-brain barrier Glutamine. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Acute liver failure [ALF] refers to the rapid loss of hepatic function, which leads to hepatocellular dysfunction and neurological impairment within days or weeks in patients without a history of liver disease [ 1 , 2 ]. These patients present a high risk of developing several life-threatening conditions such as Hepatic Encephalopathy (HE), infections, bleeding due to coagulation disorders and multi-organ failure, thus requiring specialized medical support in intensive care units [ 3 , 4 ]. HE is defined as a brain dysfunction caused by liver failure and may lead to intracranial hypertension and cerebral edema, which are the major factors of death in patients with ALF [ 5 – 7 ]. The prognosis of patients with ALF relates to HE grade. Patients with mild HE (Grades I and II) have a good chance of spontaneous recovery (around 70%), while those in advanced stages (Grades III and IV) present a much worse scenario with a recovery rate of around 20% [ 8 , 7 ]. The clinical management of ALF has improved over the last decades, but it still represents a difficult challenge to hepatologists and critical care doctors. Even with close monitoring of ALF complications and intensive supervision of clinical parameters, drastic interventions such as liver transplantation (LT) are often necessary to save the patient’s life [ 6 , 9 , 10 ]. Different clinical criteria to identify patients benefiting from LT can aid in the decision-making processes between clinical management and transplantation. Among these, the most used are the King’s College Criteria, the Clichy Criteria and MELD score [ 8 , 11 , 12 ]. These tools assess clinical characteristics of the disease (e.g., patient’s age, ALF etiology and onset of jaundice) as well as blood biomarkers (e.g., prothrombin time and serum creatinine, among others) to predict the severity of liver failure and the need for more aggressive treatment strategies. As intracranial hypertension is a central cause of coma and death in ALF, searching for biomarkers of brain impairment is relevant to assess the progression or severity of the disease [ 13 – 15 ]. Previous studies identified that ALF is associated with increased levels of amino acids in blood and CSF. Classic reports also documented high levels of amino acids in serum and CSF in several experimental models of ALF and HE [ 16 – 20 ], and recent studies performed in patients under these conditions have demonstrated similar findings [ 21 – 23 ]. A possible cause of amino acid levels increase in CSF is the altered permeability in the blood-brain barrier (BBB) caused by ALF. Studies on animal models with hyperammonemia and ALF have demonstrated BBB dysfunction [ 24 – 28 ]. High glutamine levels in CSF have been associated with the stage of HE in patients with chronic liver disease [ 29 – 33 ] and with changes in electroencephalogram (EEG) and neurological criteria in patients at intensive care units [ 34 ]. Recent data suggest that high levels of glutamine in blood refer to more severe cases and worse outcomes in patients with liver failure [ 23 ]. Our group recently published an experimental model of ALF induced by subtotal hepatectomy (surgical removal of 92% of the hepatic mass), which presented marked alterations in the EEG associated with high glutamine levels in CSF [ 35 ]. Animal models of ALF can also contribute to identify biomarkers for assessing the progression of HE. Previous studies have proposed neurological grading in rats with acute HE, in different animal models of ALF [ 36 , 37 ]. However, the criteria used to identify each neurological state are confusing and difficult to apply objectively. Like this, this study goals are: i) to propose a practical and reproducible method for evaluating the severity of HE in rats with ALF induced by subtotal hepatectomy; ii) to evaluate the levels of glutamine, amino acids and albumin in CSF as markers of hepatic encephalopathy progression; iii) to correlate BBB impairment with the modulation of these CSF markers. We suggest that amino acids and glutamine levels in CSF are potential biomarkers to identify subjects at higher risk of poor outcomes, which may contribute to the future medical management of patients with ALF. Methods Animals and experimental design The experiments were performed with 90-day-old male Wistar rats from the Central Animal House of the Department of Biochemistry, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. The animals were kept in a colony room with 12 hours of light/dark cycles (lights on 7am–7pm) and with an air conditioner in a constant temperature (22 ± 1°C). Animals had free access to water and standard commercial chow (SUPRA, Porto Alegre, RS, Brazil). The experimental protocol was approved by the Ethics Committee for Animal Research of the Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, under project number 29468, and followed the NIH Guide for the Care and Use of Laboratory Animals (NIH publication 85 − 23, revised 1996). All efforts were made to minimize the number of animals used and their suffering. Animals were divided into four different cohorts. The first cohort intended to evaluate neurological severity of HE, under observation for 72 hours, but not submitted to further interventions until the end of the experiment (Table 1 ). The second cohort intended to evaluate the plasma levels in liver function tests (Fig. 1 ) and amino acids levels (Fig. 3 ) in CSF 24 hours after surgery. The third cohort intended to evaluate Evans blue penetration into brain and albumin levels in CSF (Fig. 2 ). A fourth cohort intended to evaluate amino acids and glutamine levels in CSF 12 hours after surgery and checked for overall mortality (Fig. 4 ). Surgical procedure A subtotal hepatectomy was performed as previously described [ 38 , 35 , 39 ], with minor modifications. Anesthesia was induced and maintained with 3% isoflurane at an oxygen flow rate of 1 L/min during the whole procedure. After cleaning the abdominal wall with povidone-iodine, a median laparotomy was performed, and liver ligaments and adhesions were cut. The pedicle of the left anterior lobe was ligated and resected, followed by the right anterior lobe resection and then by the other right lobes resection. After ligation of the lobe hilum, the ischemic mass was removed from the abdominal cavity to reduce local inflammation. Both omental lobes (about 8% of liver mass) remained fully functional [ 40 ]. Lidocaine (0.8 ml) was administered intramuscularly in the wound borders after suturing the abdominal wall to reduce pain. The rats were then kept in a heated box (25°C) for 30 minutes before being returned to their home cages. Animals had free access to drinking water supplemented with 20% glucose. Additionally, 2 ml of a glucose solution (50 mg glucose/100 g of body weight) was administered i.p. at 3 different times: 0, 6 and 12 hours after surgery. Sham operations were performed following the same protocol, except for pedicle ligation and liver resection. The rats were observed every 6 hours over 72 hours to evaluate the progression of HE and mortality rate. We emphasize that this surgical ALF model has been implemented by our group in recent studies, and we have outlined data regarding blood biomarkers of liver dysfunction, brain energy metabolism as well as electroencephalographic and behavior alterations [ 35 ]. Neurological evaluation Every 6 hours, a neurological evaluation was carried out following surgical procedure – up to 72 hours. First, the cage grid was removed to stimulate animals’ locomotion (time 0). Animals presenting some spontaneous locomotion or exploratory activity (e.g., if interacting with other animals, food or cage walls), within the first 10 seconds, were considered at Grade Ø. Animals remaining immobile were tested for both righting and corneal reflexes. Righting reflex was assessed by lifting and trying to place the animal on its back on the cage floor. A normal response would be the animal turning its body to land on its feet before reaching the ground or to immediately returning to its normal position once released by the researcher. Corneal reflex was tested by softly poking the cornea with a cotton swab, which should trigger a response of eyelid closure. Animals not moving within the first 10 seconds, but presenting no abnormal reflexes were considered at Grade I. Animals showing no spontaneous locomotion and allowing to be positioned on their backs, but that quickly (< 3 seconds) returned to their normal position, were considered at Grade II. Animals presenting a significant locomotor deficit with no spontaneous locomotion or exploration, which were unable to return to their upright position when placed on their back, but which still presented normal corneal reflexes, were considered at Grade III. Comatose animals with abnormal righting reflex, corneal reflex or no response to pain stimuli were at Grade IV. Death time was registered as the last time when the animal was seen alive. As expected, all Sham-operated animals remained in stage Ø during this study. Biochemical analysis of plasma Twenty-four hours after surgery, the blood was collected from a direct cardiac puncture, using a sodium citrate tube and centrifuged at 5000 × g for 10 minutes. Plasma was stored at -80°C for subsequent measurements of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total and Direct bilirubin, Prothrombin time and Ammonia levels, using commercial kits (Labtest Diagnostica S.A., Lagoa Santa, MG, Brazil). Cerebrospinal fluid analysis CSF harvesting was performed according to previous reports from our group [ 39 , 35 , 41 ]. Animals were briefly anesthetized with 3% isoflurane and positioned in a stereotaxic frame. Samples of CSF (approximately 50 µl) were collected by direct puncture of the fourth ventricle (cisterna magna) with insulin syringes (29G). CSF was immediately centrifuged at 1000g for 10 minutes, and the supernatant was stored at -80ºC for posterior analysis. Brain sample harvesting was performed in 3 independent cohorts of animals: i) CSF samples were collected 24 hours after surgery to evaluate amino acids (Supplementary Table 1) and albumin levels; ii) whole brains were collected for evaluating Evans Blue penetration into brain (blue color); iii) CSF samples were collected 12 hours after surgery to assess the putative prognostic value of amino acid levels increase related to the risk of mortality. Amino acid levels in CSF Before analysis, CSF was extracted through a 0.22 µm filter and further deproteinized (1:5) with methanol, centrifuged at 3000 g for 10 minutes, and the supernatant was then collected. The levels of free amino acids in CSF (aspartate, glutamate, alanine, tryptophan, methionine, valine, phenylalanine, isoleucine, leucine, ornithine and lysine) as well as glutamine were determined by high-performance liquid chromatography (HPLC), according to Joseph, et al., 1986 [ 42 ]. For the analysis, we have used a reverse phase column (Supelcosil LC-18, 250 mm × 4.6 mm × 5 µm, Supelco) in a liquid chromatograph by Shimadzu Instruments (50µL loop injection valve, 40 uL injection volume) and fluorescent detection after pre-column derivatization using 100.5 µL OPA (5.4 mg OPA in 1 mL 0.2 M sodium borate pH 9.5) plus 25.5 µL 4% mercaptoethanol for 3 minutes. The mobile phase flowed at a rate of 1.4 mL/min, and the column temperature was at 25 Cº. Buffer composition was: i) 0.04 mol/L sodium dihydrogen phosphate monohydrate, pH 5.5, containing 80% of methanol; ii) 0.01 mol/L sodium dihydrogen phosphate monohydrate, pH 5.5, containing 20% of methanol. The gradient profile was modified according to the content of buffer B in the mobile phase: 100% at 0.10 min, 90% at 15 min, 48% at 10 min and 100% at 60 min. Absorbance was read at 360 nm and 455 nm, excitation and emission respectively, in a Shimadzu’s fluorescence detector. Samples of 20 µL were used, and concentration was expressed in µM (as mean ± SD). Amino acids were identified by their retention time and were quantitatively determined by using their chromatographic peak area. A known amino acid standard was used for calibration. Albumin levels in CSF We have used a high-performance liquid chromatography coupled with a fluorescence detector (HPLC-FLD) consisting of an LC Shimadzu system (Kyoto, Japan) equipped with an LC- 20AT pump, a DGU-14A degasser, thermostat set for CTO-10A column, and fluorescence detector, RF 20A to quantify albumin concentration in CSF. LC Solution software was used in data acquisition and processing. The FLD was set at 278 nm (excitation) and 335 nm (emission). Agilent ZORBAX SB-C18 reversed-phase column (5 µm particle size, 250 × 4.6 mm i.d.) was used. In this method, we implemented a gradient condition, consisting of solvent A (H2O + 0.1% formic acid) and solvent B (acetonitrile [ACN]) as follows: A → 65% B → 35% (0–5.0 min), A → 70% B → 30% (5.0–10 min), A → 65% B → 35% (10.0–17.0 min). The flow rate was set at 0.7 mL/min. FDA bioanalytical guidance complemented by the EMA was used to perform method validation. Albumin stock solutions were prepared in water at 1 mg/mL and stored at − 20 ± 2°C. For each day of analysis, standard solutions of albumin were prepared at 0.1, 0.5, 1, 10, 50 and 100 µg/mL. Sample preparation was performed by adding 10 µL liquor to the 40 µL of ACN and vortex mixed. Then, the solution was transferred to conical vials, and 10 µL were injected. Procedures followed previous guidelines and recommendations [ 43 , 44 ]. Evans Blue penetration into brain A small cohort of animals was selected to assess Evans Blue (EB) penetration into the central nervous system (CNS) parenchyma following 24 hours of surgery. EB staining indirectly accessed albumin penetration into the brain, as this molecule binds to serum albumin. Normally, neither EB nor albumin crosses the BBB. However, in some pathological conditions, albumin is able to enter the CNS carrying the EB blue stain [ 45 ]. EB (2% dissolved in saline solution) was administered by tail vein puncture (3 mL/kg) 24 hours after subtotal hepatectomy [ 46 ]. After 90 minutes, animals were anesthetized with isoflurane and euthanized by exsanguination. Brains were carefully dissected and kept on ice for image capturing. Statistical analysis The data are expressed as the mean ± S.E.M. To compare the groups, the following statistical methods were used depending on the experiments: i) One-way ANOVA followed by Tukey’s test; or ii) Student’s t-test, when mentioned, using GraphPad Prism vs. 5 (La Jolla, CA, USA). The level of significance was considered as p < 0.05 . Results Plasma levels in liver function tests following 24h of surgery Figure 1 shows that subtotal hepatectomy increased the levels of all hepatic parameters analyzed when compared to the respective Sham group ( p < 0.001) . This indicates a severe liver impairment: aspartate aminotransferase (Fig. 1 A, Sham: 28.20 ± 1.37 vs Hepatec: 66.67 ± 2.46, p < 0.001 ), alanine aminotransferase (Fig. 1 B, Sham: 33.67 ± 1.09 vs Hepatec: 71.41 ± 2.51, p < 0.001 ), total bilirubin (Fig. 1 C, Sham: 0.04 ± 0.00 vs Hepatec: 1.64 ± 0.03, p < 0.001 ), direct bilirubin (Fig. 1 D, Sham: 0.03 ± 0.00 vs Hepatec: 0.58 ± 0.04, p < 0.001 ), prothrombin time (Fig. 1 E, Sham: 12.31 ± 0.20 vs Hepatec: 25.61 ± 0.88, p < 0.001 ) and ammonia (Fig. 1 F, Sham: 23.53 ± 1.23 vs Hepatec: 61.00 ± 2.53, p < 0.001 ). Neurological HE severity following 72h of surgery Table 1 shows that subtotal hepatectomy rapidly impaired brain function. The mean onset of mild encephalopathy (Grade I) was 8.66 hours. The onset of Grade II was 16.50 hours. The mean onset of severe stages of encephalopathy was 29.65 hours (Grade III) and 33.60 hours (Grade IV). The mean time of death was 36 hours. Table 1. Neurological Evaluation of HE severity. The first column represents the neurological severity grades, the second column represents the criteria used for neurological severity classification, and the third column represents the mean onset after surgery (hours) of each neurological stage. Data are expressed as mean ± SD (hours). In the beginning, the number of rats in the experiment was n = 39. Evans Blue penetration into brain and albumin levels in CSF following 24h of surgery Figure 2 shows that albumin levels in CSF (Fig. 2 A) and the Evans Blue penetration into brain (Fig. 2 B) increased, according to the degree of neurological severity (Albumin: Sham: 1.14 ± 0.29; Grade I: 1.87 ± 0.26; Grade II: 2.41 ± 0.43; Grade III: 2.81 ± 0.03, p < 0.05 ) compared to Sham. No animal in this cohort attained Grade IV following 24 hours of surgery. Amino acid levels in CSF and HE severity correlation following 24h of surgery Figure 3 shows that the increase of amino acid levels in CSF were correlated with the Grade of HE severity (Grades I-IV). Values (Mean ± SD) are expressed in µM. Sham (Ø), Grade I, Grade II, Grade III and Grade IV, respectively: Fig. 3 A, Glutamine: 386.9 ± 109.7; 1431.0 ± 476.1; 1959.0 ± 540.8; 2586.0 ± 834.9 and 3015.0 ± 429.6, p < 0.05 ; Fig. 3 B, Glutamate: 1.25 ± 0.39; 1.46 ± 0.39; 2.29 ± 0.57; 13.06 ± 5.71 and 19.92 ± 4.35, p < 0.05 ; Fig. 3 C, Aspartate: 0.46 ± 0.19; 0.53 ± 0.34; 0.71 ± 0.32; 2.00 ± 1.45 and 4.41 ± 0.28, p < 0.05 ; Fig. 3 D, Alanine: 53.05 ± 20.9; 51.27 ± 4.81; 49.40 ± 13.26; 69.83 ± 24.10 and 43.04 ± 14.32; Fig. 3 E, Tryptophan: 2.00 ± 0.95; 3.94 ± 0.93; 2.94 ± 0.26; 6.70 ± 3.73 and 13.99 ± 6.34, p < 0.05 ; Fig. 3 F, Methionine: 3.82 ± 1.61; 14.91 ± 6.90; 15.74 ± 4.59; 32.55 ± 14.89 and 59.27 ± 19.57, p < 0.05 ; Fig. 3 G, Valine: 3.67 ± 1.83; 3.45 ± 2.79; 4.83 ± 2.13; 7.87 ± 3.25 and 12.23 ± 7.17, p < 0.05 ; Fig. 3 H, Phenylalanine: 4.43 ± 1.12; 17.54 ± 7.65; 18.14 ± 6.17; 53.42 ± 24.06 and 86.24 ± 8.72, p < 0.05 ; Fig. 3 I, Isoleucine: 1.77 ± 0.65; 2.79 ± 1.38; 2.89 ± 0.68; 5.49 ± 2.29 and 12.39 ± 0.86, p < 0.05 ; Fig. 3 J, Leucine: 3.45 ± 0.99; 3.89 ± 0.89; 5.10 ± 2.11; 8.12 ± 4.39 and 20.20 ± 5.66, p < 0.05 ; Fig. 3 K, Ornithine: 2.25 ± 1.04; 2.34 ± 0.77; 3.13 ± 0.66; 5.40 ± 2.24 and 13.58 ± 6.41, p < 0.05 ; Fig. 3 L, Lysine: 67.52 ± 19.89; 124.20 ± 41.75; 152.60 ± 45.87; 288.90 ± 108.50 and 592.50 ± 259.60, p < 0.05 . Amino acid levels in CSF related to mortality rate following 12h of surgery Figure 4 shows that the increase of amino acid levels in CSF was correlated with HE-induced mortality, pointing out that this parameter could be considered a predictor of mortality. Animals were posteriorly divided into 3 groups, and CSF samples were collected 12 hours after the surgery: i) Sham-operated group; ii) Recovered, which consisted of animals that survived the 72-hour analysis; and iii) Deceased, which consisted of animals that died between 12 hours and 72 hours after surgery. The results show that 12 hours after surgery the Recovered group did not present a significant increase of most amino acids levels in CSF, except for glutamine (Fig. 4 A, p < 0.05 ), methionine (Fig. 4 F, p < 0.05 ) and phenylalanine (Fig. 4 H, p < 0.05 ), which were mildly elevated. However, for this same period, animals in the Deceased group presented a significant increase of all amino acids levels in CSF (Fig. 4 , p < 0.05 ). The values for each amino acid (Mean ± S.D.) are expressed below in µM as Sham, Recovered and Deceased, respectively. Figure 4 A, Glutamine: 386.90 ± 109.71; 1079.01 ± 149.97 and 1928.25 ± 414.21, p < 0.05 ; Fig. 4 B, Glutamate: 1.25 ± 0.40; 1.39 ± 0.37 and 4.28 ± 3.01, p < 0.05 ; Fig. 4 C, Aspartate: 0.46 ± 0.20; 0.47 ± 0.12 and 1.19 ± 0.76, p < 0.05 ; Fig. 4 D, Alanine: 53.05 ± 20.90; 50.95 ± 13.64 and 98.48 ± 57.82, p < 0.05 ; Fig. 4 E, Tryptophan: 2.00 ± 0.94; 3.72 ± 0.43 and 8.07 ± 2.89, p < 0.05 ; Fig. 4 F, Methionine: 3.83 ± 1.61; 9.98 ± 2.45 and 23.08 ± 4.85, p < 0.05 ; Fig. 4 G, Valine: 3.68 ± 1.83; 5.70 ± 1.18 and 10.53 ± 5.94, p < 0.05 ; Fig. 4 H, Phenylalanine: 4.43 ± 1.12; 17.11 ± 2.96 and 40.84 ± 25.58, p < 0.05 ; Fig. 4 I, Isoleucine: 1.77 ± 0.65; 2.66 ± 0.26 and 4.92 ± 3.09, p < 0.05 ; Fig. 4 J, Leucine: 3.45 ± 0.99; 4.76 ± 0.76 and 10.76 ± 5.31, p < 0.05 ; Fig. 4 K, Ornithine: 2.25 ± 1.04; 2.78 ± 0.54 and 6.68 ± 3.86, p < 0.05 ; Fig. 4 L, Lysine: 67.52 ± 19.90; 137.51 ± 24.71 and 297.80 ± 130.23, p < 0.05 . Discussion This study evaluates the correlation between the neurological progression of hepatic encephalopathy (HE) and amino acids and albumin levels in the CSF of rats with acute liver failure induced by subtotal hepatectomy. In this study, our group: i) proposes a scale for a reproducible approach to the neurological evaluation of rats with ALF; ii) correlates the increase of amino acids and albumin levels in CSF with animals’ neurological status; and iii) correlates the early (12 hours) increase of amino acid levels in CSF with a prognosis of death. Based upon these results, we hypothesize that the biochemical alterations observed in this study relate to BBB impairment in animals with HE, and, therefore, we propose that the increased levels of albumin and amino acids may be a potential biomarker for future medical evaluation and management of patients with ALF. Acute liver failure is a life-threatening medical condition that frequently entails a significant brain impairment known as Hepatic Encephalopathy [ 1 , 47 , 48 ]. The reduction in hepatic workload leads to the accumulation of ammonia and other neurotoxins, which interfere with brain metabolism [ 49 , 50 ]. In our experimental model, the hepatectomized animals present a significant increase of plasma levels in biomarkers of liver dysfunctionality: AST, ALT, Total Bilirubin, Direct Bilirubin, Prothrombin time and Ammonia, in comparison with Sham rats (Fig. 1 ) as observed in patients with ALF. In humans, Hepatic Encephalopathy symptoms range from mild alterations, such as ataxia, extremity tremor, mood changes, sleep disturbances and slurred speech up to severe neurological impairment, such as muscle rigidity, hyperreflexia, obtundation and even coma [ 51 , 52 ]. Encephalopathy is clinically classified into degrees to better understand and monitor its severity [ 51 , 53 ]. Animal models of liver disease have been studied for decades to understand the pathophysiology of HE, aiming to discover new therapeutic strategies for mitigating the impacts of this condition [ 54 ]. These experimental models reproduce the various stages of the disease for observation and classification purposes to identify the progression of neurological impairment subsequently. Mans and colleagues described a neurological scale for rats with HE induced by portocaval shunting and hepatic artery ligation [ 37 ]. More recently, Cauli and colleagues described another neurological grading method for rats with ALF induced by galactosamine [ 36 ]. These neurological scales, however, describe generic signs, such as mild ataxia, mild lethargy and poor gesture control to differentiate animals from stages 0, I and II. These criteria are susceptible to observational bias and may result in different interpretations [ 55 , 37 ]. Our study proposes a modified neurological evaluation of HE, using simple and objective criteria with no additional costs or workload, which ensures better-defined criteria to sort subjects into neurological grades for HE in animal models. Our criteria are described in detail in the Methods – Neurological Evaluation section. Elevated levels of free amino acids have been documented in both serum and CSF of animals with HE [ 37 , 20 , 56 , 57 ]. These alterations seem to be consistent with elevated levels of glutamine and glutamate, which are also found in patients with HE [ 58 , 59 , 21 , 22 ]. Similarly, our group previously described elevated levels of glutamate and glutamine in CSF both in a model of hyperammonemia and of acute HE [ 41 , 35 ]. In this study, we observed a significant increase of all analyzed amino acids levels (except for alanine) in CSF in animals with ALF 24h after subtotal hepatectomy (Supplementary Table 1). These alterations were strongly correlated with the Grade of HE (Fig. 3 ). Animals with Grades I and II presented no alterations (or only mild elevations) in amino acid levels in CSF, while animals with Grades III and IV presented large increases of amino acid levels. Additionally, albumin levels in CSF were correlated with the neurological grades of HE, increasing significantly in the late stages of the disease (Fig. 2 ). Albumin presents a high molecular weight and is unable to cross the BBB under normal conditions [ 60 , 61 ]. Albumin levels in CSF only increase under pathological conditions [ 62 ]. Therefore, albumin levels in CSF are considered a marker of BBB permeability [ 63 – 65 ]. Hyperammonemia has been shown to affect BBB permeability and allows amino acids to enter into the brain [ 19 , 24 ]. Previous studies in animal models of HE and ALF indicate mild alterations in BBB and do not demonstrate a clear breakdown of this brain system [ 66 – 68 , 28 , 27 , 26 ]. Similarly, a study on patients with chronic liver disease and overt HE did not observe changes in BBB permeability, using positron emission tomography (PET) scans [ 69 ]. In the present study, however, we found elevated levels of almost all amino acids in CSF and an increase of albumin that was associated with the neurological deterioration due to HE. A diffuse penetration of various free amino acids into CSF is likely caused by a single factor, such as altered BBB permeability, rather than specific disruptions in the metabolic pathway of each amino acid caused by ammonia accumulation. This hypothesis is reinforced by the observed diffuse and homogenous increased penetration of Evans Blue staining into animals’ brain with HE, as shown in Fig. 2 B. The amino acid profile and albumin evaluation discussed above were performed 24h after subtotal hepatectomy – a result observed in previous studies carried out by our group [ 35 , 39 ]. Following 24h of surgery, most of the rats present significant neurological impairment and are not yet in the final stages of HE, as the mean time of death is after 36 hours (Table 1 ). Another cohort of animals was selected for a second experiment to harvest CSF 12h after surgery. At this period, most animals presented HE Grade I, with few animals in Grade II (Table 1 ). We consider difficult to determine, via neurological parameters, which animals will improve their clinical condition and which will progress to coma and death. However, the evaluation of amino acid levels in CSF 12h after surgery allowed us to make a clear distinction between these two groups: the first group (Recovered group) presented a normal or mild elevation in amino acid levels and survived up to 72h (final period of the evaluation); while the second group (Deceased group) presented a marked elevation of amino acid levels in CSF (Fig. 4 ). The glutamatergic system is believed to have an important role in ammonia detoxification in the brain, specifically via the production of glutamine [ 50 , 35 ]. However, it is also believed that the accumulation of glutamine in the brain may cause dangerous effects, because of its involvement in the pathogenesis of brain edema and may act as a Trojan Horse by releasing ammonium into the mitochondria of the cells, thus disturbing brain bioenergetics [ 70 – 72 ]. Elevated levels of glutamine in serum and CSF have been associated with neurological decline as evidenced by EEG and with poor clinical outcomes in patients with HE [ 34 , 23 ]. In this experiment, the evaluation of amino acid levels in CSF was of great value in differentiating healthy animals from those with HE, and animals with poor prognoses from those spontaneously recovered (Fig. 4 ). Considering this fact, elevated glutamine after 12 hours differentiated rats with HE from Sham-operated animals. Glutamine levels also allowed us to differentiate, with greater accuracy, the animals with favorable prognoses from those that would die before the end of the experiment (Fig. 4 A). This was also true for methionine and phenylalanine (Figs. 4 F and 4 H, respectively). It is important to emphasize that the sample collection was performed 12 hours after surgery, a point at which it was not possible to predict the animals’ outcome evaluation by neurological grade of HE. These results correlate the increase of amino acids levels in CSF to an important outcome, such as mortality rate. We assume that if we could observe this same pattern of amino acid increased levels in CSF in humans with ALF, elevated glutamine levels could potentially be a useful biomarker for evaluating these patients. Conclusion This study presents a neurological grading protocol for evaluating the progression of acute HE in rats with ALF and describes an increase of amino acids and albumin levels in the CSF of these animals. The CSF alterations correlate with the severity of neurological symptoms and with the mortality rate. Based on the literature and on our results, we suggest that the increase of amino acid levels and glutamine and albumin levels in CSF may be due to impaired BBB activity. To the best of our knowledge, this is the first study that associates BBB permeability markers with a neurological evaluation under experimental ALF. We also believe that our data offer important information regarding the potential role of amino acids and glutamine in detecting, even in the early stages of HE, the subjects that will progress to coma and death. Therefore, we hypothesize that these molecules have the potential to be useful biomarkers for monitoring and evaluating the progression of HE in humans. Declarations Funding This work was supported by Brazilian agencies and grants: CNPq, CAPES, FAPERGS, Brazilian Institute of Neuroscience/FINEP, INCT/Excitotoxicity and Neuroprotection (465671/2014-4). The authors declare no competing financial interests. Competing Interests The authors declare they have no financial interests. Authors Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Pedro Arend Guazzelli, Felipe dos Santos Fachim, Anderson Santos Travassos, Caroline Casagrande Schaukoski, Pâmela Cristina Lukasewicz Ferreira and Fernanda Uruth Fontella. The final version of the manuscript was written by Pedro Arend Guazzelli, Adriano Martimbianco de Assis and Diogo Onofre Souza, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethics approval The experimental protocol was approved by the Ethics Committee for Animal Research of the Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, under project number 29468, and followed the NIH Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1996). All efforts were made to minimize the number of animals used and their suffering. References Bunchorntavakul C, Reddy KR (2017) Acute Liver Failure. Clinics in liver disease 21. 769–792. 10.1016/j.cld.2017.06.002 . 4 Bernal W, Auzinger G, Dhawan A, Wendon J (2010) Acute liver failure. Lancet 376(9736):190–201. doi: 10.1016/S0140-6736(10)60274-7 Jayalakshmi VT, Bernal W (2020) Update on the management of acute liver failure. 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Hepatology 44(4):788–794. doi: 10.1002/hep.21357 Supplementary Files SupplementaryTable1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2093824","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":141561758,"identity":"56007af6-d552-4fa0-b494-fd4566d2ac0a","order_by":0,"name":"Pedro Arend Guazzelli","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul Instituto de Ciências Básicas da Saúde: Universidade Federal do Rio Grande do Sul Instituto de Ciencias Basicas da 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(A) \u003c/strong\u003eAspartate aminotransferase (U/L); \u003cstrong\u003e(B) \u003c/strong\u003eAlanine aminotransferase (U/L); \u003cstrong\u003e(C)\u003c/strong\u003e Total Bilirubin (mg/dL); \u003cstrong\u003e(D)\u003c/strong\u003e Direct Bilirubin (mg/dL); \u003cstrong\u003e(E) \u003c/strong\u003eProthrombin time (s); and \u003cstrong\u003e(F)\u003c/strong\u003e Ammonia (µmol/L). The results are expressed as the mean ± S.E.M. *** (\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e) indicates a significant difference from the Sham group (Students t-test). n: Sham=15, Hepatectomy=15.\u003c/p\u003e","description":"","filename":"Figure1NEW.png","url":"https://assets-eu.researchsquare.com/files/rs-2093824/v1/ede30875d778a87335a993ac.png"},{"id":27393846,"identity":"b668460b-5943-46b5-b8ae-59d06580b8e8","added_by":"auto","created_at":"2022-10-05 19:59:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":230406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between Evans Blue penetration into brain/albumin levels in CSF and HE severity 24h after surgery. \u003c/strong\u003eThe hepatectomy groups were divided according to the neurological stage at the time of the sample collection. \u003cstrong\u003e(A) \u003c/strong\u003eAlbumin levels in CSF:\u003cstrong\u003e \u003c/strong\u003eSham (n=9); Grade I (n=6); Grade II (n=5); Grade III (n=3). No animal in this cohort reached Grade IV after 24 hours. *(\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e) indicates a significant difference from all other groups (One-way ANOVA followed by Tukey’s test); \u003cstrong\u003e(B)\u003c/strong\u003e representative images of the animals’ brains 90 minutes after IV injection of Evans Blue.\u003c/p\u003e","description":"","filename":"Figure2NEW.png","url":"https://assets-eu.researchsquare.com/files/rs-2093824/v1/3def15b9cdd28d08d0158cee.png"},{"id":27394069,"identity":"ec844098-102e-4a35-852a-b8d959e9f95f","added_by":"auto","created_at":"2022-10-05 20:04:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31055,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between amino acids/glutamine levels in CSF and HE severity 24h after surgery. (A)\u003c/strong\u003e Glutamine; \u003cstrong\u003e(B)\u003c/strong\u003e Glutamate; \u003cstrong\u003e(C)\u003c/strong\u003e Aspartate; \u003cstrong\u003e(D)\u003c/strong\u003e Alanine; \u003cstrong\u003e(E)\u003c/strong\u003e Tryptophan; \u003cstrong\u003e(F) \u003c/strong\u003eMethionine; \u003cstrong\u003e(G)\u003c/strong\u003e Valine; \u003cstrong\u003e(H)\u003c/strong\u003e Phenylalanine; \u003cstrong\u003e(I)\u003c/strong\u003e Isoleucine; \u003cstrong\u003e(J)\u003c/strong\u003e Leucine; \u003cstrong\u003e(K)\u003c/strong\u003e Ornithine; and \u003cstrong\u003e(L)\u003c/strong\u003e Lysine. Data are expressed as mean ± SD (µM). Sham (n=14), Grade I (n=6), Grade II (n=6), Grade III (n=4), Grade IV (n=3). *(\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e) indicates a significant difference from all other groups (One-way ANOVA followed by Tukey’s test).\u003c/p\u003e","description":"","filename":"Figure3NEW.png","url":"https://assets-eu.researchsquare.com/files/rs-2093824/v1/bd511107dc2bdfd55a071d31.png"},{"id":27393848,"identity":"cee1420f-0166-436b-b58e-6eaa7abe21fd","added_by":"auto","created_at":"2022-10-05 19:59:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":35903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between amino acids/glutamine levels in CSF and mortality rate 72h after surgery. (A)\u003c/strong\u003e Glutamine; \u003cstrong\u003e(B)\u003c/strong\u003eGlutamate; \u003cstrong\u003e(C)\u003c/strong\u003e Aspartate; \u003cstrong\u003e(D)\u003c/strong\u003e Alanine; \u003cstrong\u003e(E)\u003c/strong\u003e Tryptophan; \u003cstrong\u003e(F) \u003c/strong\u003eMethionine; \u003cstrong\u003e(G)\u003c/strong\u003e Valine; \u003cstrong\u003e(H)\u003c/strong\u003e Phenylalanine; \u003cstrong\u003e(I)\u003c/strong\u003eIsoleucine; \u003cstrong\u003e(J)\u003c/strong\u003eLeucine; \u003cstrong\u003e(K)\u003c/strong\u003e Ornithine; and \u003cstrong\u003e(L)\u003c/strong\u003e Lysine.\u003cstrong\u003e \u003c/strong\u003eData represent amino acids levels in CSF 12 hours after subtotal hepatectomy of the following groups: Sham (n=14), Recovered (animals that survived 72 hours after surgery) (n=8) and Deceased (animals that died from 12 to 72 hours after surgery) (n=9). Data are expressed as mean ± SD (µM). *\u003cem\u003e(p\u0026lt;0.05\u003c/em\u003e) indicates a significant difference from all other groups (One-way ANOVA followed by Tukey’s test).\u003c/p\u003e","description":"","filename":"Figure4NEW.png","url":"https://assets-eu.researchsquare.com/files/rs-2093824/v1/f8b76ff15fee6e7ac641687f.png"},{"id":27906786,"identity":"a76cbcd5-9db7-4b4a-8fba-02f327fd5ca3","added_by":"auto","created_at":"2022-10-18 05:49:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1002845,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2093824/v1/6d890485-156e-4478-a4ff-348747d3db87.pdf"},{"id":27393845,"identity":"c372800f-1117-41de-9b0d-3127f5cece20","added_by":"auto","created_at":"2022-10-05 19:59:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":738070,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-2093824/v1/377270decece5ba899f6753b.docx"}],"financialInterests":"","formattedTitle":"Blood-brain barrier permeability and increased levels of amino acids in cerebrospinal fluid are associated with brain alterations in rats with acute liver failure","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute liver failure [ALF] refers to the rapid loss of hepatic function, which leads to hepatocellular dysfunction and neurological impairment within days or weeks in patients without a history of liver disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These patients present a high risk of developing several life-threatening conditions such as Hepatic Encephalopathy (HE), infections, bleeding due to coagulation disorders and multi-organ failure, thus requiring specialized medical support in intensive care units [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. HE is defined as a brain dysfunction caused by liver failure and may lead to intracranial hypertension and cerebral edema, which are the major factors of death in patients with ALF [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The prognosis of patients with ALF relates to HE grade. Patients with mild HE (Grades I and II) have a good chance of spontaneous recovery (around 70%), while those in advanced stages (Grades III and IV) present a much worse scenario with a recovery rate of around 20% [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe clinical management of ALF has improved over the last decades, but it still represents a difficult challenge to hepatologists and critical care doctors. Even with close monitoring of ALF complications and intensive supervision of clinical parameters, drastic interventions such as liver transplantation (LT) are often necessary to save the patient\u0026rsquo;s life [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Different clinical criteria to identify patients benefiting from LT can aid in the decision-making processes between clinical management and transplantation. Among these, the most used are the King\u0026rsquo;s College Criteria, the Clichy Criteria and MELD score [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These tools assess clinical characteristics of the disease (e.g., patient\u0026rsquo;s age, ALF etiology and onset of jaundice) as well as blood biomarkers (e.g., prothrombin time and serum creatinine, among others) to predict the severity of liver failure and the need for more aggressive treatment strategies. As intracranial hypertension is a central cause of coma and death in ALF, searching for biomarkers of brain impairment is relevant to assess the progression or severity of the disease [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies identified that ALF is associated with increased levels of amino acids in blood and CSF. Classic reports also documented high levels of amino acids in serum and CSF in several experimental models of ALF and HE [\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and recent studies performed in patients under these conditions have demonstrated similar findings [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A possible cause of amino acid levels increase in CSF is the altered permeability in the blood-brain barrier (BBB) caused by ALF. Studies on animal models with hyperammonemia and ALF have demonstrated BBB dysfunction [\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHigh glutamine levels in CSF have been associated with the stage of HE in patients with chronic liver disease [\u003cspan additionalcitationids=\"CR30 CR31 CR32\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and with changes in electroencephalogram (EEG) and neurological criteria in patients at intensive care units [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Recent data suggest that high levels of glutamine in blood refer to more severe cases and worse outcomes in patients with liver failure [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Our group recently published an experimental model of ALF induced by subtotal hepatectomy (surgical removal of 92% of the hepatic mass), which presented marked alterations in the EEG associated with high glutamine levels in CSF [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnimal models of ALF can also contribute to identify biomarkers for assessing the progression of HE. Previous studies have proposed neurological grading in rats with acute HE, in different animal models of ALF [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, the criteria used to identify each neurological state are confusing and difficult to apply objectively. Like this, this study goals are: i) to propose a practical and reproducible method for evaluating the severity of HE in rats with ALF induced by subtotal hepatectomy; ii) to evaluate the levels of glutamine, amino acids and albumin in CSF as markers of hepatic encephalopathy progression; iii) to correlate BBB impairment with the modulation of these CSF markers. We suggest that amino acids and glutamine levels in CSF are potential biomarkers to identify subjects at higher risk of poor outcomes, which may contribute to the future medical management of patients with ALF.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eAnimals and experimental design\u003c/h2\u003e\n\u003cp\u003eThe experiments were performed with 90-day-old male Wistar rats from the Central Animal House of the Department of Biochemistry, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. The animals were kept in a colony room with 12 hours of light/dark cycles (lights on 7am\u0026ndash;7pm) and with an air conditioner in a constant temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C). Animals had free access to water and standard commercial chow (SUPRA, Porto Alegre, RS, Brazil). The experimental protocol was approved by the Ethics Committee for Animal Research of the Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, under project number 29468, and followed the NIH Guide for the Care and Use of Laboratory Animals (NIH publication 85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996). All efforts were made to minimize the number of animals used and their suffering.\u003c/p\u003e\n\u003cp\u003eAnimals were divided into four different cohorts. The first cohort intended to evaluate neurological severity of HE, under observation for 72 hours, but not submitted to further interventions until the end of the experiment (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The second cohort intended to evaluate the plasma levels in liver function tests (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and amino acids levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) in CSF 24 hours after surgery. The third cohort intended to evaluate Evans blue penetration into brain and albumin levels in CSF (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). A fourth cohort intended to evaluate amino acids and glutamine levels in CSF 12 hours after surgery and checked for overall mortality (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eSurgical procedure\u003c/h2\u003e\n\u003cp\u003eA subtotal hepatectomy was performed as previously described [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e], with minor modifications. Anesthesia was induced and maintained with 3% isoflurane at an oxygen flow rate of 1 L/min during the whole procedure. After cleaning the abdominal wall with povidone-iodine, a median laparotomy was performed, and liver ligaments and adhesions were cut. The pedicle of the left anterior lobe was ligated and resected, followed by the right anterior lobe resection and then by the other right lobes resection. After ligation of the lobe hilum, the ischemic mass was removed from the abdominal cavity to reduce local inflammation. Both omental lobes (about 8% of liver mass) remained fully functional [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eLidocaine (0.8 ml) was administered intramuscularly in the wound borders after suturing the abdominal wall to reduce pain. The rats were then kept in a heated box (25\u0026deg;C) for 30 minutes before being returned to their home cages. Animals had free access to drinking water supplemented with 20% glucose. Additionally, 2 ml of a glucose solution (50 mg glucose/100 g of body weight) was administered i.p. at 3 different times: 0, 6 and 12 hours after surgery. Sham operations were performed following the same protocol, except for pedicle ligation and liver resection. The rats were observed every 6 hours over 72 hours to evaluate the progression of HE and mortality rate.\u003c/p\u003e\n\u003cp\u003eWe emphasize that this surgical ALF model has been implemented by our group in recent studies, and we have outlined data regarding blood biomarkers of liver dysfunction, brain energy metabolism as well as electroencephalographic and behavior alterations [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eNeurological evaluation\u003c/h2\u003e\n\u003cp\u003eEvery 6 hours, a neurological evaluation was carried out following surgical procedure \u0026ndash; up to 72 hours. First, the cage grid was removed to stimulate animals\u0026rsquo; locomotion (time 0). Animals presenting some spontaneous locomotion or exploratory activity (e.g., if interacting with other animals, food or cage walls), within the first 10 seconds, were considered at Grade \u0026Oslash;. Animals remaining immobile were tested for both righting and corneal reflexes. Righting reflex was assessed by lifting and trying to place the animal on its back on the cage floor. A normal response would be the animal turning its body to land on its feet before reaching the ground or to immediately returning to its normal position once released by the researcher. Corneal reflex was tested by softly poking the cornea with a cotton swab, which should trigger a response of eyelid closure. Animals not moving within the first 10 seconds, but presenting no abnormal reflexes were considered at Grade I. Animals showing no spontaneous locomotion and allowing to be positioned on their backs, but that quickly (\u0026lt;\u0026thinsp;3 seconds) returned to their normal position, were considered at Grade II. Animals presenting a significant locomotor deficit with no spontaneous locomotion or exploration, which were unable to return to their upright position when placed on their back, but which still presented normal corneal reflexes, were considered at Grade III. Comatose animals with abnormal righting reflex, corneal reflex or no response to pain stimuli were at Grade IV. Death time was registered as the last time when the animal was seen alive. As expected, all Sham-operated animals remained in stage \u0026Oslash; during this study.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eBiochemical analysis of plasma\u003c/h2\u003e\n\u003cp\u003eTwenty-four hours after surgery, the blood was collected from a direct cardiac puncture, using a sodium citrate tube and centrifuged at 5000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 minutes. Plasma was stored at -80\u0026deg;C for subsequent measurements of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total and Direct bilirubin, Prothrombin time and Ammonia levels, using commercial kits (Labtest Diagnostica S.A., Lagoa Santa, MG, Brazil).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eCerebrospinal fluid analysis\u003c/h2\u003e\n\u003cp\u003eCSF harvesting was performed according to previous reports from our group [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. Animals were briefly anesthetized with 3% isoflurane and positioned in a stereotaxic frame. Samples of CSF (approximately 50 \u0026micro;l) were collected by direct puncture of the fourth ventricle (cisterna magna) with insulin syringes (29G). CSF was immediately centrifuged at 1000g for 10 minutes, and the supernatant was stored at -80\u0026ordm;C for posterior analysis. Brain sample harvesting was performed in 3 independent cohorts of animals: i) CSF samples were collected 24 hours after surgery to evaluate amino acids (Supplementary Table\u0026nbsp;1) and albumin levels; ii) whole brains were collected for evaluating Evans Blue penetration into brain (blue color); iii) CSF samples were collected 12 hours after surgery to assess the putative prognostic value of amino acid levels increase related to the risk of mortality.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eAmino acid levels in CSF\u003c/h2\u003e\n\u003cp\u003eBefore analysis, CSF was extracted through a 0.22 \u0026micro;m filter and further deproteinized (1:5) with methanol, centrifuged at 3000 g for 10 minutes, and the supernatant was then collected. The levels of free amino acids in CSF (aspartate, glutamate, alanine, tryptophan, methionine, valine, phenylalanine, isoleucine, leucine, ornithine and lysine) as well as glutamine were determined by high-performance liquid chromatography (HPLC), according to Joseph, et al., 1986 [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. For the analysis, we have used a reverse phase column (Supelcosil LC-18, 250 mm \u0026times; 4.6 mm \u0026times; 5 \u0026micro;m, Supelco) in a liquid chromatograph by Shimadzu Instruments (50\u0026micro;L loop injection valve, 40 uL injection volume) and fluorescent detection after pre-column derivatization using 100.5 \u0026micro;L OPA (5.4 mg OPA in 1 mL 0.2 M sodium borate pH 9.5) plus 25.5 \u0026micro;L 4% mercaptoethanol for 3 minutes. The mobile phase flowed at a rate of 1.4 mL/min, and the column temperature was at 25 C\u0026ordm;. Buffer composition was: i) 0.04 mol/L sodium dihydrogen phosphate monohydrate, pH 5.5, containing 80% of methanol; ii) 0.01 mol/L sodium dihydrogen phosphate monohydrate, pH 5.5, containing 20% of methanol. The gradient profile was modified according to the content of buffer B in the mobile phase: 100% at 0.10 min, 90% at 15 min, 48% at 10 min and 100% at 60 min. Absorbance was read at 360 nm and 455 nm, excitation and emission respectively, in a Shimadzu\u0026rsquo;s fluorescence detector. Samples of 20 \u0026micro;L were used, and concentration was expressed in \u0026micro;M (as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). Amino acids were identified by their retention time and were quantitatively determined by using their chromatographic peak area. A known amino acid standard was used for calibration.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eAlbumin levels in CSF\u003c/h2\u003e\n\u003cp\u003eWe have used a high-performance liquid chromatography coupled with a fluorescence detector (HPLC-FLD) consisting of an LC Shimadzu system (Kyoto, Japan) equipped with an LC- 20AT pump, a DGU-14A degasser, thermostat set for CTO-10A column, and fluorescence detector, RF 20A to quantify albumin concentration in CSF. LC Solution software was used in data acquisition and processing. The FLD was set at 278 nm (excitation) and 335 nm (emission). Agilent ZORBAX SB-C18 reversed-phase column (5 \u0026micro;m particle size, 250 \u0026times; 4.6 mm i.d.) was used. In this method, we implemented a gradient condition, consisting of solvent A (H2O\u0026thinsp;+\u0026thinsp;0.1% formic acid) and solvent B (acetonitrile [ACN]) as follows: A \u0026rarr; 65% B \u0026rarr; 35% (0\u0026ndash;5.0 min), A \u0026rarr; 70% B \u0026rarr; 30% (5.0\u0026ndash;10 min), A \u0026rarr; 65% B \u0026rarr; 35% (10.0\u0026ndash;17.0 min). The flow rate was set at 0.7 mL/min. FDA bioanalytical guidance complemented by the EMA was used to perform method validation. Albumin stock solutions were prepared in water at 1 mg/mL and stored at \u0026minus;\u0026thinsp;20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. For each day of analysis, standard solutions of albumin were prepared at 0.1, 0.5, 1, 10, 50 and 100 \u0026micro;g/mL. Sample preparation was performed by adding 10 \u0026micro;L liquor to the 40 \u0026micro;L of ACN and vortex mixed. Then, the solution was transferred to conical vials, and 10 \u0026micro;L were injected. Procedures followed previous guidelines and recommendations [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eEvans Blue penetration into brain\u003c/h2\u003e\n\u003cp\u003eA small cohort of animals was selected to assess Evans Blue (EB) penetration into the central nervous system (CNS) parenchyma following 24 hours of surgery. EB staining indirectly accessed albumin penetration into the brain, as this molecule binds to serum albumin. Normally, neither EB nor albumin crosses the BBB. However, in some pathological conditions, albumin is able to enter the CNS carrying the EB blue stain [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. EB (2% dissolved in saline solution) was administered by tail vein puncture (3 mL/kg) 24 hours after subtotal hepatectomy [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. After 90 minutes, animals were anesthetized with isoflurane and euthanized by exsanguination. Brains were carefully dissected and kept on ice for image capturing.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eThe data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. To compare the groups, the following statistical methods were used depending on the experiments: i) One-way ANOVA followed by Tukey\u0026rsquo;s test; or ii) Student\u0026rsquo;s t-test, when mentioned, using GraphPad Prism vs. 5 (La Jolla, CA, USA). The level of significance was considered as \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlasma levels in liver function tests following 24h of surgery\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows that subtotal hepatectomy increased the levels of all hepatic parameters analyzed when compared to the respective Sham group (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/em\u003e. This indicates a severe liver impairment: aspartate aminotransferase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Sham: 28.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37 vs Hepatec: 66.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), alanine aminotransferase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Sham: 33.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09 vs Hepatec: 71.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.51, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), total bilirubin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Sham: 0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 vs Hepatec: 1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), direct bilirubin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Sham: 0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 vs Hepatec: 0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), prothrombin time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, Sham: 12.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 vs Hepatec: 25.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) and ammonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, Sham: 23.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23 vs Hepatec: 61.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eNeurological HE severity following 72h of surgery\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows that subtotal hepatectomy rapidly impaired brain function. The mean onset of mild encephalopathy (Grade I) was 8.66 hours. The onset of Grade II was 16.50 hours. The mean onset of severe stages of encephalopathy was 29.65 hours (Grade III) and 33.60 hours (Grade IV). The mean time of death was 36 hours.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e\u003cstrong\u003eTable 1. Neurological Evaluation of HE severity.\u0026nbsp;\u003c/strong\u003eThe first column represents the neurological severity grades, the second column represents the criteria used for neurological severity classification, and the third column represents the mean onset after surgery (hours) of\u0026nbsp;each neurological stage. Data are expressed as mean ± SD (hours). In the beginning, the number of rats in the experiment was n = 39.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cimg 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\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEvans Blue penetration into brain and albumin levels in CSF following 24h of surgery\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that albumin levels in CSF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and the Evans Blue penetration into brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) increased, according to the degree of neurological severity (Albumin: Sham: 1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29; Grade I: 1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26; Grade II: 2.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43; Grade III: 2.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) compared to Sham. No animal in this cohort attained Grade IV following 24 hours of surgery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAmino acid levels in CSF and HE severity correlation following 24h of surgery\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the increase of amino acid levels in CSF were correlated with the Grade of HE severity (Grades I-IV). Values (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) are expressed in \u0026micro;M. Sham (\u0026Oslash;), Grade I, Grade II, Grade III and Grade IV, respectively: Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Glutamine: 386.9\u0026thinsp;\u0026plusmn;\u0026thinsp;109.7; 1431.0\u0026thinsp;\u0026plusmn;\u0026thinsp;476.1; 1959.0\u0026thinsp;\u0026plusmn;\u0026thinsp;540.8; 2586.0\u0026thinsp;\u0026plusmn;\u0026thinsp;834.9 and 3015.0\u0026thinsp;\u0026plusmn;\u0026thinsp;429.6, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Glutamate: 1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39; 1.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39; 2.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57; 13.06\u0026thinsp;\u0026plusmn;\u0026thinsp;5.71 and 19.92\u0026thinsp;\u0026plusmn;\u0026thinsp;4.35, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Aspartate: 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19; 0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34; 0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32; 2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45 and 4.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, Alanine: 53.05\u0026thinsp;\u0026plusmn;\u0026thinsp;20.9; 51.27\u0026thinsp;\u0026plusmn;\u0026thinsp;4.81; 49.40\u0026thinsp;\u0026plusmn;\u0026thinsp;13.26; 69.83\u0026thinsp;\u0026plusmn;\u0026thinsp;24.10 and 43.04\u0026thinsp;\u0026plusmn;\u0026thinsp;14.32; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, Tryptophan: 2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95; 3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93; 2.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26; 6.70\u0026thinsp;\u0026plusmn;\u0026thinsp;3.73 and 13.99\u0026thinsp;\u0026plusmn;\u0026thinsp;6.34, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, Methionine: 3.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61; 14.91\u0026thinsp;\u0026plusmn;\u0026thinsp;6.90; 15.74\u0026thinsp;\u0026plusmn;\u0026thinsp;4.59; 32.55\u0026thinsp;\u0026plusmn;\u0026thinsp;14.89 and 59.27\u0026thinsp;\u0026plusmn;\u0026thinsp;19.57, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, Valine: 3.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83; 3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.79; 4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13; 7.87\u0026thinsp;\u0026plusmn;\u0026thinsp;3.25 and 12.23\u0026thinsp;\u0026plusmn;\u0026thinsp;7.17, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, Phenylalanine: 4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12; 17.54\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65; 18.14\u0026thinsp;\u0026plusmn;\u0026thinsp;6.17; 53.42\u0026thinsp;\u0026plusmn;\u0026thinsp;24.06 and 86.24\u0026thinsp;\u0026plusmn;\u0026thinsp;8.72, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, Isoleucine: 1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65; 2.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38; 2.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68; 5.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29 and 12.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, Leucine: 3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99; 3.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89; 5.10\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11; 8.12\u0026thinsp;\u0026plusmn;\u0026thinsp;4.39 and 20.20\u0026thinsp;\u0026plusmn;\u0026thinsp;5.66, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK, Ornithine: 2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04; 2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77; 3.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66; 5.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24 and 13.58\u0026thinsp;\u0026plusmn;\u0026thinsp;6.41, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL, Lysine: 67.52\u0026thinsp;\u0026plusmn;\u0026thinsp;19.89; 124.20\u0026thinsp;\u0026plusmn;\u0026thinsp;41.75; 152.60\u0026thinsp;\u0026plusmn;\u0026thinsp;45.87; 288.90\u0026thinsp;\u0026plusmn;\u0026thinsp;108.50 and 592.50\u0026thinsp;\u0026plusmn;\u0026thinsp;259.60, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAmino acid levels in CSF related to mortality rate following 12h of surgery\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows that the increase of amino acid levels in CSF was correlated with HE-induced mortality, pointing out that this parameter could be considered a predictor of mortality. Animals were posteriorly divided into 3 groups, and CSF samples were collected 12 hours after the surgery: i) Sham-operated group; ii) Recovered, which consisted of animals that survived the 72-hour analysis; and iii) Deceased, which consisted of animals that died between 12 hours and 72 hours after surgery. The results show that 12 hours after surgery the Recovered group did not present a significant increase of most amino acids levels in CSF, except for glutamine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, p\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), methionine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, p\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and phenylalanine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, p\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), which were mildly elevated. However, for this same period, animals in the Deceased group presented a significant increase of all amino acids levels in CSF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, p\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The values for each amino acid (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D.) are expressed below in \u0026micro;M as Sham, Recovered and Deceased, respectively. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Glutamine: 386.90\u0026thinsp;\u0026plusmn;\u0026thinsp;109.71; 1079.01\u0026thinsp;\u0026plusmn;\u0026thinsp;149.97 and 1928.25\u0026thinsp;\u0026plusmn;\u0026thinsp;414.21, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Glutamate: 1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40; 1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 and 4.28\u0026thinsp;\u0026plusmn;\u0026thinsp;3.01, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Aspartate: 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20; 0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 and 1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, Alanine: 53.05\u0026thinsp;\u0026plusmn;\u0026thinsp;20.90; 50.95\u0026thinsp;\u0026plusmn;\u0026thinsp;13.64 and 98.48\u0026thinsp;\u0026plusmn;\u0026thinsp;57.82, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, Tryptophan: 2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94; 3.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 and 8.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, Methionine: 3.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61; 9.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45 and 23.08\u0026thinsp;\u0026plusmn;\u0026thinsp;4.85, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, Valine: 3.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83; 5.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18 and 10.53\u0026thinsp;\u0026plusmn;\u0026thinsp;5.94, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, Phenylalanine: 4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12; 17.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96 and 40.84\u0026thinsp;\u0026plusmn;\u0026thinsp;25.58, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, Isoleucine: 1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65; 2.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 and 4.92\u0026thinsp;\u0026plusmn;\u0026thinsp;3.09, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, Leucine: 3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99; 4.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 and 10.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.31, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, Ornithine: 2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04; 2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 and 6.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL, Lysine: 67.52\u0026thinsp;\u0026plusmn;\u0026thinsp;19.90; 137.51\u0026thinsp;\u0026plusmn;\u0026thinsp;24.71 and 297.80\u0026thinsp;\u0026plusmn;\u0026thinsp;130.23, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study evaluates the correlation between the neurological progression of hepatic encephalopathy (HE) and amino acids and albumin levels in the CSF of rats with acute liver failure induced by subtotal hepatectomy. In this study, our group: i) proposes a scale for a reproducible approach to the neurological evaluation of rats with ALF; ii) correlates the increase of amino acids and albumin levels in CSF with animals\u0026rsquo; neurological status; and iii) correlates the early (12 hours) increase of amino acid levels in CSF with a prognosis of death. Based upon these results, we hypothesize that the biochemical alterations observed in this study relate to BBB impairment in animals with HE, and, therefore, we propose that the increased levels of albumin and amino acids may be a potential biomarker for future medical evaluation and management of patients with ALF.\u003c/p\u003e \u003cp\u003eAcute liver failure is a life-threatening medical condition that frequently entails a significant brain impairment known as Hepatic Encephalopathy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The reduction in hepatic workload leads to the accumulation of ammonia and other neurotoxins, which interfere with brain metabolism [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In our experimental model, the hepatectomized animals present a significant increase of plasma levels in biomarkers of liver dysfunctionality: AST, ALT, Total Bilirubin, Direct Bilirubin, Prothrombin time and Ammonia, in comparison with Sham rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) as observed in patients with ALF. In humans, Hepatic Encephalopathy symptoms range from mild alterations, such as ataxia, extremity tremor, mood changes, sleep disturbances and slurred speech up to severe neurological impairment, such as muscle rigidity, hyperreflexia, obtundation and even coma [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Encephalopathy is clinically classified into degrees to better understand and monitor its severity [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnimal models of liver disease have been studied for decades to understand the pathophysiology of HE, aiming to discover new therapeutic strategies for mitigating the impacts of this condition [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. These experimental models reproduce the various stages of the disease for observation and classification purposes to identify the progression of neurological impairment subsequently. Mans and colleagues described a neurological scale for rats with HE induced by portocaval shunting and hepatic artery ligation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. More recently, Cauli and colleagues described another neurological grading method for rats with ALF induced by galactosamine [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These neurological scales, however, describe generic signs, such as mild ataxia, mild lethargy and poor gesture control to differentiate animals from stages 0, I and II. These criteria are susceptible to observational bias and may result in different interpretations [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our study proposes a modified neurological evaluation of HE, using simple and objective criteria with no additional costs or workload, which ensures better-defined criteria to sort subjects into neurological grades for HE in animal models. Our criteria are described in detail in the Methods \u0026ndash; \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003eNeurological Evaluation\u003c/span\u003e section.\u003c/p\u003e \u003cp\u003eElevated levels of free amino acids have been documented in both serum and CSF of animals with HE [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. These alterations seem to be consistent with elevated levels of glutamine and glutamate, which are also found in patients with HE [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Similarly, our group previously described elevated levels of glutamate and glutamine in CSF both in a model of hyperammonemia and of acute HE [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, we observed a significant increase of all analyzed amino acids levels (except for alanine) in CSF in animals with ALF 24h after subtotal hepatectomy (Supplementary Table\u0026nbsp;1). These alterations were strongly correlated with the Grade of HE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Animals with Grades I and II presented no alterations (or only mild elevations) in amino acid levels in CSF, while animals with Grades III and IV presented large increases of amino acid levels.\u003c/p\u003e \u003cp\u003eAdditionally, albumin levels in CSF were correlated with the neurological grades of HE, increasing significantly in the late stages of the disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Albumin presents a high molecular weight and is unable to cross the BBB under normal conditions [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Albumin levels in CSF only increase under pathological conditions [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Therefore, albumin levels in CSF are considered a marker of BBB permeability [\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Hyperammonemia has been shown to affect BBB permeability and allows amino acids to enter into the brain [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Previous studies in animal models of HE and ALF indicate mild alterations in BBB and do not demonstrate a clear breakdown of this brain system [\u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Similarly, a study on patients with chronic liver disease and overt HE did not observe changes in BBB permeability, using positron emission tomography (PET) scans [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. In the present study, however, we found elevated levels of almost all amino acids in CSF and an increase of albumin that was associated with the neurological deterioration due to HE. A diffuse penetration of various free amino acids into CSF is likely caused by a single factor, such as altered BBB permeability, rather than specific disruptions in the metabolic pathway of each amino acid caused by ammonia accumulation. This hypothesis is reinforced by the observed diffuse and homogenous increased penetration of Evans Blue staining into animals\u0026rsquo; brain with HE, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003eThe amino acid profile and albumin evaluation discussed above were performed 24h after subtotal hepatectomy \u0026ndash; a result observed in previous studies carried out by our group [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Following 24h of surgery, most of the rats present significant neurological impairment and are not yet in the final stages of HE, as the mean time of death is after 36 hours (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Another cohort of animals was selected for a second experiment to harvest CSF 12h after surgery. At this period, most animals presented HE Grade I, with few animals in Grade II (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We consider difficult to determine, via neurological parameters, which animals will improve their clinical condition and which will progress to coma and death. However, the evaluation of amino acid levels in CSF 12h after surgery allowed us to make a clear distinction between these two groups: the first group (Recovered group) presented a normal or mild elevation in amino acid levels and survived up to 72h (final period of the evaluation); while the second group (Deceased group) presented a marked elevation of amino acid levels in CSF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe glutamatergic system is believed to have an important role in ammonia detoxification in the brain, specifically via the production of glutamine [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, it is also believed that the accumulation of glutamine in the brain may cause dangerous effects, because of its involvement in the pathogenesis of brain edema and may act as a Trojan Horse by releasing ammonium into the mitochondria of the cells, thus disturbing brain bioenergetics [\u003cspan additionalcitationids=\"CR71\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Elevated levels of glutamine in serum and CSF have been associated with neurological decline as evidenced by EEG and with poor clinical outcomes in patients with HE [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this experiment, the evaluation of amino acid levels in CSF was of great value in differentiating healthy animals from those with HE, and animals with poor prognoses from those spontaneously recovered (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Considering this fact, elevated glutamine after 12 hours differentiated rats with HE from Sham-operated animals. Glutamine levels also allowed us to differentiate, with greater accuracy, the animals with favorable prognoses from those that would die before the end of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This was also true for methionine and phenylalanine (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, respectively). It is important to emphasize that the sample collection was performed 12 hours after surgery, a point at which it was not possible to predict the animals\u0026rsquo; outcome evaluation by neurological grade of HE. These results correlate the increase of amino acids levels in CSF to an important outcome, such as mortality rate. We assume that if we could observe this same pattern of amino acid increased levels in CSF in humans with ALF, elevated glutamine levels could potentially be a useful biomarker for evaluating these patients.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents a neurological grading protocol for evaluating the progression of acute HE in rats with ALF and describes an increase of amino acids and albumin levels in the CSF of these animals. The CSF alterations correlate with the severity of neurological symptoms and with the mortality rate. Based on the literature and on our results, we suggest that the increase of amino acid levels and glutamine and albumin levels in CSF may be due to impaired BBB activity. To the best of our knowledge, this is the first study that associates BBB permeability markers with a neurological evaluation under experimental ALF. We also believe that our data offer important information regarding the potential role of amino acids and glutamine in detecting, even in the early stages of HE, the subjects that will progress to coma and death. Therefore, we hypothesize that these molecules have the potential to be useful biomarkers for monitoring and evaluating the progression of HE in humans.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Brazilian agencies and grants: CNPq, CAPES, FAPERGS, Brazilian Institute of Neuroscience/FINEP, INCT/Excitotoxicity and Neuroprotection (465671/2014-4). The authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by\u0026nbsp;Pedro Arend Guazzelli,\u0026nbsp;Felipe dos Santos Fachim, Anderson Santos Travassos, Caroline Casagrande Schaukoski, P\u0026acirc;mela Cristina Lukasewicz Ferreira and Fernanda Uruth Fontella. The final version of the manuscript was written by\u0026nbsp;Pedro Arend Guazzelli,\u0026nbsp;Adriano Martimbianco de Assis and Diogo Onofre Souza,\u0026nbsp;and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocol was approved by the Ethics Committee for Animal Research of the Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, under project number 29468, and followed the NIH Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1996). 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Hepatology 44(4):788\u0026ndash;794. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/hep.21357\u003c/span\u003e\u003cspan address=\"10.1002/hep.21357\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Acute liver failure, Hepatic encephalopathy, Cerebrospinal Fluid, Amino Acids, Albumin, Blood-brain barrier, Glutamine.","lastPublishedDoi":"10.21203/rs.3.rs-2093824/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2093824/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcute liver failure (ALF) is a life-threatening medical condition that often leads to hepatic encephalopathy (HE). Animals and humans with HE have shown elevated cerebrospinal fluid (CSF) levels of glutamine and glutamate, which may be associated with brain impairment. In this study, we aim to evaluate the relationship between blood-brain barrier (BBB) integrity and CSF amino acid levels with the neurological status of rats after subtotal hepatectomy. Adult male Wistar rats underwent a subtotal hepatectomy (removing 92% of hepatic mass or SHAM group) and were divided into 4 (four) cohorts. Animals with ALF presented severe neurological impairment and high mortality rates when compared to the SHAM group (Cohort 1). We performed a hepatic function test 24 hours after subtotal hepatectomy, which demonstrated a significant increase of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total Bilirubin, Direct Bilirubin, Prothrombin time and Ammonia levels in blood (Cohort 2). Additionally, the increase of amino acids, glutamine, and albumin levels in CSF as well as of Evans Blue penetration into the brain tissue was correlated with the neurological grades of HE, indicating signs of impaired BBB permeability induced by ALF (Cohort 3). Furthermore, the animals\u0026rsquo; mortality rate showed a positive correlation with the increase of amino acid levels in CSF following subtotal hepatectomy (after 12h, Cohort 4). Our data highlight the potential role of amino acid levels in CSF, especially glutamine, for detection purposes even in the early stages of HE and suggest that these molecules offer prognostic value for patients progressing to coma and death.\u003c/p\u003e","manuscriptTitle":"Blood-brain barrier permeability and increased levels of amino acids in cerebrospinal fluid are associated with brain alterations in rats with acute liver failure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-05 19:58:59","doi":"10.21203/rs.3.rs-2093824/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4b59aa4d-8115-4061-82bc-56c7748aaac7","owner":[],"postedDate":"October 5th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-10-18T05:48:52+00:00","versionOfRecord":[],"versionCreatedAt":"2022-10-05 19:58:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2093824","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2093824","identity":"rs-2093824","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}